The T cell-mediated response to complex antigens involves recognition of selected peptide epitopes presented in the context of MHC molecules expressed on antigen presenting cells. The choice of these immunogenic epitopes from among the often hundreds or thousands of amino acids comprising an antigenic protein depends significantly on the binding properties of a given MHC type and the interactions of specific amino acids with a T cell receptor. Understanding which peptide epitopes participate in T cell-mediated immunity provides a basis for directed modulation of the immune response, including development of peptide vaccines and therapies against allergens, autoimmune diseases and tumors. (See, e.g., Chang et al., J. Immunol. 162:1156 (1999); Rolland and O'Hehir, Curr. Opin. Immunol. 10:640 (1998); Wicker et al., J. Clin. Invest. 98:2597 (1996); Falk et al., J. Exp. Med. 191:717 (2000); Wang and Rosenberg, Immunol. Rev. 170:85 (1999).)
The standard approach for cloning T cells and mapping epitopes of an antigen involves antigen challenge of peripheral blood mononuclear cells (PBMC) followed by plating individual cells into 96-well plates. Cells are then expanded and assayed for MHC restriction and peptide specificities by screening clones with individual peptides which cover the antigen, a labor intensive and time consuming process. Epitopes can also be identified using a combination of chromatography and mass spectroscopy to identify peptides bound to MHC molecules, an approach which requires purification of MHC molecules and coupling to a receptor. Alternatively, epitopes can be identified using a recently described flow cytometry-based approach that utilizes Interferon gamma (IFNγ) production as a marker of reactivity. While this approach simplifies isolation of epitope-specific clones, the task of identifying individual MHC restriction elements remains.
A number of recent studies have employed soluble MHC multimers to directly identify T cells restricted to specific peptide epitopes. This technology has been utilized to track T cells specific for both viral antigens (see, e.g., Altman et al., Science 274:94 (1996); Callan et al., J. Exp. Med. 187:1395 (1998); Gallimore et al., J. Exp. Med. 187:1383 (1998); Wilson et al., J. Exp. Med. 188:785 (1998)) and tumor antigens (Lee et al., Nat. Med. 5:677 (1999); Pittet et al., J. Exp. Med. 190:705 (1999); Dunbar et al., J. Immunol. 162:6959 (1999); Molldrem et al., Cancer Res. 59:2675 (1999)) in both animal models and in humans when the peptide epitope is known.
The majority of these studies have focused on class I restricted T cells, because efforts in producing MHC class II molecules are hampered by difficulties in generating stable soluble forms of the MHC class II molecules and inefficiency in forming multimers of these molecules. In particular, stable soluble MHC class II molecules bound to peptide have been difficult to form from human MHC class II molecules. Human MHC class II molecules have been difficult to load with peptide, and the resulting multimers can be unstable.
A related problem with using MHC class II molecules to identify epitopes in antigens is that peptides are screened individually to identify the epitope(s) of the antigen. Thus, elucidation of specific epitopes from complex antigens can be a cumbersome and difficult process as it generally involves extensive phenotype screening of T cell clones isolated from whole-antigen stimulated cells.
Thus, there is a need for methods for efficiently screening human T cells to identify MHC class II epitopes within an antigen. The present invention satisfies this and other needs.